Selenophosphate synthetase forms the energy-rich compound, selenophosphate, from ATP and selenide. An essential cysteine residue in the E. coli enzyme is replaced by selenocysteine, threonine, or arginine in various eukaryotic forms of the enzyme. The relative catalytic activities of these replacements have been evaluated. Free selenide is inactive as substrate in vitro for the threonine- and arginine-substituted enzymes. Also, the relatively low activities of the S and Se enzymes on selenide indicated that another form of selenium should be the in vivo substrate. Sulfur transferases that normally deliver active sulfur from enzyme persulfide intermediates also can serve as selenium delivery systems in vitro, thus replacing free selenide, which is very toxic. However, known selenium-specific enzymes, such as L-selenocysteine lyase, should function in vivo. Initial purification steps for selenocysteine lyases and also selenophosphate synthetase isoenzymes from two potentially rich anaerobic bacterial sources were developed. The selenophosphate synthetase from Methanococcus vannielii shows significant sequence differences from known forms of the enzyme. Mechanistic studies on wild type and mutant forms of selenophosphate synthetase from E. coli show that phosphorylation of enzyme by ATP is unaffected. Further attempts to delineate the phosphorylation site are in progress. Three forms of the enzyme that were decreased in mass by removal of C-terminal peptides of varying lengths were produced by mutagenesis. In view of the observed correlations of selenium deficiency and low glutathione levels with poor survival of HIV-infected individuals, it was of interest to study selenium metabolism in T cells. Synthesis of 75-Se-labeled selenoproteins by human Jurkat T cells was investigated and the profiles of labeled proteins from HIV-infected and uninfected T cells were compared.